Nitric oxide (NO), released at endothelial level, plays an essential role in the regulation of vessel tone and is considered an essential molecule in the regulation of cardiovascular system. It has been recently hypothesized that NO, besides having a paracrine effect, is also capable of acting under an endocrine mechanism, inducing a vessel-active action in zones that are remote from the biosynthesis/delivering site.
In particular, it is supposed that high doses of NO, delivered via inhalation, can be beneficial not only at pulmonary level, but also at systemic level, and that an intravenous infusion of a NO solution can cause vasodilation.
Owing to the brief half life of NO in the hematic area (about 2 ms), the endocrine effect of NO is supposed to be mediated by steady and bioactive carrying mechanisms, capable of guiding NO from biosynthesis delivery sites of to ischemic/hipoxic zones. Presently different forms of deposition and transport of NO at hematic level have been observe: S-nitrosocysteine, S-nitrosoglutathione (defined as low molecular weight nitrosothiols, LMW-RSNOs), as well as S-nitrosoalbumin and S-nitrosohemoglobin (defined as high molecular weigth nitrosothiols, HMW-RSNOs), for which the radical R represents an aminoacid, a polypeptide and a proteine respectively. Such compounds are capable of releasing NO.
The presence at hematic level of these forms of deposition of NO, whose levels increase in a significant way after inhalation of NO or by supplying NO-donor drugs, has been confirmed by many researches in different animal models and in humans (as described by Cannon et al. above cited). However their actual contribution in conservative NO metabolisms have still to be defined, as well as their task and their relevance in physiological and pathological conditions.
It has also been supposed that some NO molecules are displaced by nitrosylhemoglobin to the residual of cysteine 93 of hemoglobin beta chain, forming S-nitroso-hemoglobin (SNO-Hb) that could act as vasodilator (Gladwin M T, Schechter AN Circ Res. 2004,94,851).
Some authors (Deem S Free Radic Biol Med. 2004,36, 698-706) hypothesize that, at peripheral level, when Hb releases oxygen to the tissues, SNO-Hb is capable of conveying and releasing NO in bioactive form.
In addition to the involvement of NO and of its derivatives in the cardiovascular system, many authors have described other effects of nitric oxide, among which the following can be cited: nitric oxide and S-nitrosothiols have antimicrobial effects (De Groote M A Fang F C Clin Infect Dis. 1995, Suppl. 2, S162-5), inhibit platelet aggregation (Hirayama A, Noronha-Dutra A A, Gordge M P, Neild GH, Hothersall J S Nitric Oxide, 1999, 3, 95-104), are bronchodilators (Bannenberg G, Xue J, Engman L, Cotgreave I, Moldeus P, Ryrfeldt A J Drugl Exp Ther, 1995, 272, 1238-45), inhibit the intestinal motility (Slivka A, Chuttani R, Carr-Locke DL, Kobzik L, Bredt D S, Loscalzo J, Stamler J S. J Clin Invest. 1994, 94, 1792-8) and are involved in different regulation processes of the central nervous system, such as nhibition of lipidic peroxidation and of oxidative damage (Rauhala P, Lin A M, Chiueh C C. FASEB J. 1998, 12, 165-73), as well as of peripheral nervous system and of the immunologic system.
Therefore, endogenous and/or esogenous compounds capable of releasing NO, known as NO-donor molecules, in the body are of high scientific interest.
In particular, S-nitrosoglutathione (GSNO), present in plasma and in lung and brain extracellular fluids, has different biological effects. GSNO is capable of, for example, relaxing the smooth muscle of the respiratory system, increasing the ciliary motility , to inhibit transfer of amiloride-sensitive sodium in epithelium aerial ducts.
A specific use of S-nitrosothiols, and, in particular, of GSNO, is described in WO95/07691. It teaches the therapeutic or prophylactic use of GSNO, for treatment of thrombosis of damaged vascular areas.
In EP 412699, instead, the use is described of S-nitrosothiols as therapeutic agent for cardiovascular diseases, in particular against hypertension, and for treatment of angina pectoris.
The steadiness of S-nitrosothiols depends on different factors, in particular, the characteristics of radical R, heat, light, the presence of ions of transition metals, the presence of other thiols, etc. Therefore, for fully exploiting the therapeutic and diagnostic potentiality it is necessary to study some biochemical, physiological and pharmacological aspects still not much known.
To this end it is relevant the ability to determine the amount of S-nitrosothiols in biological fluids (blood, plasma, saliva, urine, pulmonary fluid, liquor, amniotic fluid, etc.) through simple, rapide, precise and accurate techniques.
The methods for determining S-nitrosothiols (RSNO) in biological fluids can be classified as:
(i) Direct methods for RSNOs.
a) UV Spectrophotometry at 334 nm; owing to the low molar absorption coefficient (ε=977 M-1 cm-1) of the S-nitroso unit, the detection by means of UV Spectrophotometry after chromatographic separation or electrophoresis (Capillary Zone Electrophoresis, CZE) limits its detection at micromolar level).
b) Electrochemical detectors (detection limit=1 μM)
(ii) Indirect methods, based on the decomposition of RSNOs according to the reactions:
RSNO→RSH/RSSR+NO
NO→nitrites/nitrates and on the detection of their metabolites:
a) NO;
b) nitrites;
c) reduced thiol(RSH)).
The decomposition of the S-nitrosocysteinyl group is presently carried out through photolysis or chemical reduction.
NO is revealed normally by means of chemiluminescence or electrochemical detector of Clark type or of planar amperometric type (detection limit=1 μM) or by means of electron spin resonance spectroscopy (EPR).
Chemiluminescence is based on the reaction of NO radical with ozone according to the reaction: NO•+O3→NO2+O2γ NO•+O3+hν.
The EPR involves the entrapment of NO• by a complex with an eme-proteine that form a nitrosyl-eme-proteine. Both chemiluminescence and EPR are very sensitive techniques, but require dedicated measuring systems.
Nitrites can be revealed by means of various techniques like Griess reaction, fluorimetry and electrochemical detection coupled or not to liquid chromatography or to gas chromatography with mass spectrometry detector.
Chromatographic techniques and, in particular, high performance liquid chromatography (HPLC), allow separating two or more compounds present in a solvent exploiting the affinity balance between a “steady phase” located in a chromatographic column and a “mobile phase” that flows through the column same. The principle at the basis of this technique is that a substance more affine to the steady phase with respect to the mobile phase takes a longer time to cover the chromatographic column with respect to a substance with low affinity to the steady phase and high affinity for mobile phase. The sample to analyse is injected at the beginning of the chromatographic column where it is “pushed” through the steady phase by the mobile phase under high pressures. To obtain a effective separation it is necessary that the size of the filling particles is very low, an for this reason is necessary applying a high pressure if has to be maintained a reasonable flow rate of the eluent and then a suitable time of analysis. At the end of the column a detector is mounted (fluorimetric or spectrophotometric or eletrochemical detector), and a computer for quantifying and/or checking the injected substances.
However, the HPLC apparatus have very high costs and each single test requires very long time.
Some attempts have been made for determining S-nitrosothiols, like in WO 2006007403, where it is detected and measured the S-nitrosothiolic bond in cells and molecules containing the eme group.
Instead, in WO9710493 and U.S. Pat. No. 5,891,735 a method is described for measuring nitrosyl Fe(II)-hemoglobin, whereas WO9820336 relates to a method for detection of nitric oxide (NO) by means of EPR spectroscopy in fluids through the reactivity of NO with N-methyl-D-glucamine dithiocarbamate (MGD), a low molecular weight chelating compound, containing sulphide, complexed with iron ions [(MGD)2-Fe] Finally, in WO0216934 a test for S-nitrosothiols is described by means of EPR.
From the above it is apparent that the measurement of metabolytes of S-nitrosothiols (NO or nitrites) in biological fluids is difficult, and requires long and complex procedures of treatment of the samples, as well as equipment specialized. It is also known that the choice of the method for preparing the sample and the systems used to break the S-NO bond (photolysis, HgCl2, HgCl2/V(III), KI/I2, Cys/KI/Cu(I), Cu(I)/Cys, Cu(I)/KI/I2, CO/Cu(I)/Cys, DTT) represent a critical point for the purpose of obtaining reliable, precise and accurate analytical data.
Finally, the methods described above are aspecific, i.e. give a measurement of total RSNO present in the sample, but a differentiation (specification) of the only S-nitrosothiols can be carried out only through the use of chromatographic techniques.
It is also known that a higher difficulty associated with computing RSNO in biological fluids and, in particular, in plasma, is the physiological presence in such matrix of anti-oxidant and anti-free radical systems (uric acid, ascorbic acid, vitamines and, in particular, B12 vitamine). It has been hypothesized that whichever the mechanism is used to break the S—NO bond, once freed the NO radical, in the presence of such anti-oxidant and anti-free radical systems, the NO radical is quickly decomposed and not more revealable by fluorescent, or chemiluminescent probes, commonly used for detection of NO.
Owing to these difficulties, the different analytic approaches used, for example for determining RSNO in plasma, have given values that span over three orders of magnitude, from nanomolar concentration levels to micromolar concentration levels.